Holoprosencephaly (HPE) is a developmental defect of the forebrain, which occurs in 1 out of 250 pregnancies. HPE is characterized by either forebrain truncation or failure of forebrain septation and various other midline defects. In the developing mouse embryo, the forebrain is first specified during gastrulation by the anterior visceral endoderm (AVE), an extra-embryonic tissue. The AVE specifies forebrain in the underlying embryonic tissue by blocking the signals from the primitive streak that provide posterior character to the tissue. As development proceeds, the AVE is displaced by the anterior definitive endoderm (ADE), which also has forebrain specification activity. Cripto, which is expressed in the posterior aspect of the gastrulating embryo, is critically involved in AVE migration and ADE specification. Cripto null embryos have severe gastrulation defects, whereas Cripto hypomorphs have HPE. I have isolated a mouse line, gonzo, that also displays gastrulation and HPE phenotypes. Furthermore, I have mapped the gene responsible for this mutation to a small region of chromosome 1, and have identified a mutation in Pig-N, a GPI-biosynthesis enzyme. Because the phenotypes of Cripto and gonzo mutants are strikingly similar, and because Cripto is a GPI-anchored protein, I will test the following hypotheses: First, HPE results from either an AVE migration defect or failure to specify the ADE. Second, the Pig-N mutation is responsible for the HPE phenotype. Third, aberrant GPI modification of Cripto disrupts Cripto signaling - and is the causative factor for the HPE phenotype. In this proposal I will complete the following aims: Aim 1: I will further characterize the HPE phenotype, and specifically look at specification of the AVE and ADE by examining molecular markers known to be involved in head morphogenesis. Aim 2: I will confirm that the mutation in Pig-N is responsible for the HPE phenotype and determine its effect on Pig-N stability, localization and activity. Aim 3: I will determine Cripto activity in gonzo embryos, and will attempt to rescue the phenotype by artificially targeting Cripto to its site of activation. Holoprosencephaly (which occurs in 1 out of 250 pregnancies) is characterized by forebrain defects;the most severe cases result in a single, cyclopic eye. I have identified a new gene in the mouse that results in holoprosencephaly when it is mutated. This proposal will identify the normal function of this gene thus leading to a better understanding of the causes of holoprosencephaly.